Project Summary Atherosclerosis, the main underlying cause of death worldwide, is characterized by chronic inflammation and accumulation of lipids in the arterial wall. Excessive lipid accumulation by macrophages (M?s) and vascular smooth muscle cells (SMCs) plays a key role in the initiation and progression of atherosclerosis. It is generally accepted that atherosclerosis arise from LDL modification in the arterial wall and its subsequent internalization by M?s and SMCs through a variety of scavenger receptors. The role of scavenger receptor-independent lipid uptake in atherosclerosis, the physiological factors stimulating this process, and the signaling mechanisms involved remain poorly characterized. We reported that matrix protein thrombospondin-1 (TSP1) stimulates direct, scavenger receptor-independent uptake of unmodified, native LDL (nLDL) in M?s. TSP1 via its cognate receptor CD47 activates actin-binding protein cofilin, leading to macropinocytosis of nLDL, and excessive cholesterol accumulation. The signaling mechanisms downstream of CD47 that stimulate macropinocytosis are unknown. Although phospholipase C (PLC) and slingshot phosphatase 1 (SSH1) have been shown to activate cofilin, their roles in TSP1-induced macropinocytosis have not yet been investigated. Moreover, the ability of TSP1-CD47 signaling to stimulate M? macropinocytosis in atherosclerotic vessels in vivo and the significance of lipid macropinocytosis in the pathogenesis of atherosclerosis remain to be determined. Our novel preliminary data show that TSP1 and CD47 knockout mice are protected from atherosclerosis and the macropinocytosis inhibitor EIPA decreases atherosclerotic lesion formation in hypercholesterolemic mice. We hypothesize that TSP1 via CD47 promotes lipid macropinocytosis in the arterial wall, contributing to lipid accumulation and the pathogenesis of atherosclerosis. The hypothesis will be tested via the following aims: (1) examining for the first time whether CD47 receptor signaling in M?s stimulates macropinocytosis via PLC- and SSH1-mediated cofilin activation and contributes to atherosclerosis development; (2) exploring whether M?s internalize lipoproteins via macropinocytosis in atherosclerotic arteries in vivo and that deletion of NHE1 (major target of EIPA) specifically in M?s attenuates atherosclerosis, and (3) investigating whether TSP1 binding to CD47 stimulates macropinocytosis in M?-like SMCs via Nox1-mediated cofilin activation. The proposal will employ global and cell-specific knockout mice, and other genetic tools to test the hypothesis. Specific targeting of CD47 via multiple approaches (antibody blockade, siRNA/morpholino silencing, and CD47 activating peptide sequences) will provide confirmation of results obtained in genetic mutants. Multiple complementary techniques will be used to study macropinocytosis in vitro (pharmacological, genetic, fluorescence/high-resolution microscopy) and in vivo (cofilin mutants, AngioSPARK 680, M?-specific NHE1 knockouts). This innovative proposal has the potential to reveal important new mechanisms of lipid internalization and provide a paradigm shift in our knowledge about how atherosclerosis develops.